Membrane-adherent self-assembled systems for treatment of ocular disorders

ABSTRACT

A liquid crystalline drug delivery system for ocular administration. The drug delivery system, which is mucoadhesive, biocompatible, non-irritating, and tissue permeable, contains nanoparticles stably dispersed in an aqueous solution and can be formulated for sustained release. Also provided are methods for producing the drug delivery system and methods for treating ocular disorders by administering it to a subject.

RELATED APPLIUCATIONS

This application is a continuation of U.S. application Ser. No.15/308,000, filed on Oct. 31, 2016, which claims priority toInternational Application No. PCT/US2015/028748, filed on May 1, 2015,which claims priority to U.S. Provisional Application No. 61/987,012,filed on May 1, 2014. The contents of all prior applications areincorporated herein by reference in their entirety.

BACKGROUND Field

The application relates to nanostructured dispersions that can be usedto effectively treat disorders and diseases of the eye by administeringthe dispersions on the ocular surface, in the anterior chamber, and inthe posterior chamber.

Background Information

Many ophthalmic formulations include drug crystals suspended inointments which are composed of mineral oil and petrolatum. Suchformulations often result in irritation of the eye and patientnon-compliance due to blurry vision and inconvenience. Other ophthalmicformulations are eye-drops containing drug suspensions in an aqueoussolution, some of them viscous to extend residence time on the ocularsurface.

The effective use of eye-drops is limited by the fact that manytherapeutically valuable agents cause local irritation when topicallydosed to the eye. The cornea is highly sensitive to the application ofchemical agents. As such, this sensitivity significantly limits the useof many otherwise valuable therapeutic agents.

Another issue with existing ocular drug formulations is poorbioavailability of the drug. For example, poorly soluble drugs deliveredto the front of the eye as a suspension, e.g., eye drops, must dissolveprior to being absorbed into the eye by diffusion. Problematically, therate of drug dissolution is typically much slower than the rate of fluidclearance from the ocular surface. Thus, ineffective drug absorption,i.e., poor bioavailability, is one of the issues that confoundsfront-of-the-eye drug delivery.

To address this issue, insoluble or poorly soluble drugs, e.g.,prostaglandins and difluprednate, are typically dissolved in an organicexcipient followed by emulsification in an aqueous vehicle. The use ofemulsions frequently leads to irritation of the ocular surface,resulting from the use of excipients that cause ocular surfaceinflammation. This is especially true when the medication is utilizedfor chronic ocular surface disease therapies, such as therapies forglaucoma, dry eye, and allergies.

Further, emulsions are inherently unstable, resulting in coalescence andsubsequent separation of the phases.

The issues are different for back-of-the-eye diseases. For example, drugsuspensions of triamcinolone acetonide have been injected intravitreallyto alleviate inflammation resulting from diabetic macular edema.Multiple injections into the posterior segments of the eye can causeendophthalmitis and gradual retinal detachment.

The need exists for formulations for ocular administration which arenon-irritating, stable, and capable of delivering a drug at therapeuticconcentrations for an extended period. Additionally, a sustained releasedelivery system that is non-toxic and membrane-mimetic is needed for thetreatment of back-of-the-eye diseases.

SUMMARY

To meet the needs discussed above, a liquid crystalline drug deliverysystem is provided. The system contains nanoparticles having a size of40-900 nm dispersed in an aqueous solution. The nanoparticles include alipidic component and an alcohol. The aqueous solution contains amucoadhesive hydrophilic polymer and a buffer.

The lipidic component is present at 0.1-1% by weight of the system andthe alcohol is present at 0.1-5% by weight of the system. Themucoadhesive hydrophilic polymer is present at 1-5% by weight of thesystem.

A method for producing the liquid crystalline drug delivery system isalso disclosed. The method includes the following steps: (i) forming afirst solution containing a lipidic component and an alcohol, the firstsolution being maintained at a first temperature; (ii) obtaining asecond solution that includes a mucoadhesive hydrophilic polymer and abuffer, the second solution being aqueous and maintained at a secondtemperature; (iii) mixing the first solution and the second solution toform a combined nano/micro-dispersion, the mixing accomplished by a highenergy mixing process; (iv) subjecting the combinednano/micro-dispersion to microfluidization at a third temperature toform a nano-dispersion; and (v) incubating the nano-dispersion at 2-5°C. to form a liquid crystalline drug delivery system.

In the method disclosed above, the first solution and the secondsolution are mixed at a weight ratio of 1:1 to 1:15.

Additionally provided is a liquid crystalline drug delivery systemproduced by the above-described method.

Further, a method for treating an ocular disorder in a subject isdisclosed including the steps of identifying a subject having an oculardisorder and administering to an eye of the subject the liquidcrystalline drug delivery system described above.

In another aspect, the use of the liquid crystalline drug deliverysystem in the manufacture of a medicament for treating ocular disordersis disclosed.

The details of one or more embodiments of the invention are set forth inthe drawings and description below. Other features, objects, andadvantages of the invention will be apparent from the description andfrom the claims. The contents of all documents cited herein are herebyincorporated by reference in their entirety.

DETAILED DESCRIPTION

As mentioned above, the liquid crystalline drug delivery system includesnanoparticles dispersed in an aqueous solution. The delivery system hasa unique internal morphology that contains poorly soluble drug moleculesdissolved in its interstices for sustained release and absorption,resulting in greater bioavailability than is possible with suspensions.Both hydrophilic and hydrophobic drugs can be incorporated into theliquid crystalline delivery system, either individually or incombination.

The delivery system is a biphasic liquid, with a nanoparticle phasecontained with a continuous aqueous phase. The drug is dissolved in thenanoparticle phase, which contains both hydrophilic and hydrophobiccomponents. When mixed together, the phases interact with one another toform a liquid crystalline phase. The interaction between thenanoparticle phase and the continuous phase display the uniquecharacteristics of an ordered nanostructured assembly. The phasesseparately are not ordered or liquid crystalline.

A liquid crystalline phase is defined as a state of matter havingproperties between a conventional liquid and a solid crystal. A liquidcrystal may flow like a liquid, but its molecules may exist incrystal-like orientations. A liquid crystalline phase, when viewed underan optical microscope under crossed polarizers, will displaymulticolored textures, i.e., birefringence. Liquid crystalline phasesalso have distinct melt transitions when heated, as determined bydifferential scanning calorimetry X-ray diffraction techniques cane alsoused to characterize liquid crystals, due to the ability of crystals todisplay the Bragg reflection of light.

The liquid crystalline drug delivery system contains nanoparticles withsizes from 40 nm-900 nm, which can easily permeate through all tissuesof the eye, such the cornea and the sclera. The delivery system isdesigned to be mucoadhesive, to enhance residence time on the ocularsurface.

As mentioned above, the liquid crystalline drug delivery system includesnanoparticles dispersed in an aqueous solution. As also mentioned above,the nanoparticles include a lipidic component and an alcohol. Thelipidic component can include, e.g., phosphatidylcholine and mediumchain triglycerides. The alcohol can be cetyl alcohol.

The nanoparticles can also include one or more of cholesterol, glycerol,polyethylene glycol (PEG) 400, polypropylene glycol (PPG), PEG-stearate,poloxamer 407, tyloxapol, polysorbate 80, castor oil, and PEGylatedcastor oil. Additionally, polymers such as poly(lactic-co-glycolic acid)(PLGA) can be included in the nanoparticles for sustained releaseformulations.

In one embodiment, the nanoparticles include phosphatidylcholine, mediumchain triglycerides, and cetyl alcohol. In another embodiment, thenanoparticles include phosphatidylcholine, medium chain triglycerides,cholesterol, and cetyl alcohol.

The aqueous solution mentioned above contains a mucoadhesive hydrophilicpolymer. The polymer can be, but is not limited to, sodium hyaluronate,xanthan gum, guar gum, carboxymethylcellulose, albumin,hydroxypropylcellulose, polyethylene glycol, polyethyleneimine, mucin,1-4 beta glucan, poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide), tamarind seed polysaccharide, sodiumalginate, polycarbopol, and polycarbophil, or derivatives and mixturesthereof.

The aqueous solution also contains a buffer. The buffer can be, but isnot limited to, sodium acetate, sodium dihydrogen phosphate, disodiumhydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogenphosphate, ε-aminocaproic acid; amino acid salts such as sodiumglutamate, boric acid, and citric acid. Preferably, the buffer is aphosphate buffer.

In a particular embodiment of the liquid crystalline drug deliverysystem, the nanoparticles include phosphatidylcholine and medium chaintriglycerides, cetyl alcohol, PEG-400, PPG, PEG-stearate, poloxamer 407,tyloxapol, polysorbate 80, and castor oil, and the aqueous solutioncontains sodium hyaluronan. In another particular embodiment, thenanoparticles include phosphatidylcholine and medium chaintriglycerides, cholesterol, cetyl alcohol, PEG-400, PPG, PEG-stearate,poloxamer 407, tyloxapol, polysorbate 80, and castor oil, and theaqueous solution contains sodium hyaluronan, sodium phosphate dibasic,and sodium phosphate monobasic.

The components mentioned above can be included in the liquid crystallinedrug delivery system in the following amounts, expressed as weight % ofthe system: 0.1-1% phosphatidylcholine and medium chain triglycerides;0.1-5% cetyl alcohol; 0.2-2% PEG-400; 0.2-1% PPG; 0.1-0.7% PEG-stearate;0.1-0.25% poloxamer 407; 0.01-0.15% tyloxapol; 0.01-0.02% polysorbate80; 1-5% castor oil; 0.1-0.5% sodium hyaluronate; 0.01-0.02% sodiumphosphate monobasic; 0.05% sodium phosphate dibasic, and 75-90%deionized water (dH₂O). An alternative liquid crystalline drug deliverysystem contains all of these components and also contains 0.02-0.2%cholesterol. A further embodiment includes 0.1-0.5% xanthan gum insteadof the sodium hyaluronate. An additional embodiment contains 0.1-0.5%carboxymethylcellulose instead of the sodium hyaluronan.

In a specific embodiment, the liquid crystalline drug delivery systemincludes, by weight, 1% phosphatidylcholine and medium chaintriglycerides, 1% cetyl alcohol, 2% PEG-400, 1% PPG, 1%, 0.7%PEG-stearate, 0.22% poloxamer 407, 0.10% tyloxapol, 0.10% polysorbate80, and 3.8% castor oil, 0.14% sodium hyaluronate, 0.02% sodiumphosphate monobasic; 0.05% sodium phosphate dibasic, and the balancedH₂O. An alternative liquid crystalline drug delivery system containsthe same amounts of all these components and also contains 0.2%cholesterol.

The liquid crystalline drug delivery system described above can alsocontain an active pharmaceutical ingredient (API) at 0.01-0.5% by weightof the system. The API is loaded in the nanoparticles. The API can be,but is not limited to, fluticasone propionate, dexamethasone,betamethasone, budesonide, triamcinolone acetonide, methyl prednisolone,cortisone, beclometasone, fluticasone furoate, deoxycorticosteroneacetate, loteprednol etabonate, difluprednate, fluorometholone,rimexolone, travoprost, moxifloxacin, prednisolone acetate,posaconazole, budesonide, netilmycin, or mupirocin.

In particular embodiments, the liquid crystalline drug delivery systemincludes, by weight, 0.01-0.1% fluticasone propionate, 0.01-0.1%dexamethasone, 0.01-0.1% difluprednate, 0.1-0.5% loteprednol etabonate,0.1-0.5% posaconazole, 0.1-0.5% budesonide, 0.05-0.5% netilmycin, or0.05-0.5% mupirocin.

In a specific embodiment, the liquid crystalline drug delivery systemcontains 0.1% by weight fluticasone propionate.

Each of the above-described embodiments of the liquid crystalline drugdelivery system can have a pH of 6-7.5, an osmolarity of 250-340 mOsm/L,and a viscosity of 200-1000 cP.

The liquid crystalline drug delivery system described above is storagestable. For example, the nanoparticles dispersed in the aqueous solutiondo not settle out of the dispersion for at least 90 days.

The liquid crystalline drug delivery system can be a spray, aninjectable, or formulated as eye drops. The liquid crystalline drugdelivery system can be a high-viscosity liquid preloaded into a syringe.

The liquid crystalline drug delivery system disclosed herein can beproduced by the following steps.

Initially, a first solution containing a lipidic component and analcohol is formed. The lipidic component can include phosphatidylcholineand medium chain triglycerides in a preferred embodiment. The alcoholcan be cetyl alcohol. After forming the solution, it can be maintainedat 40-55° C. In an alternative embodiment, an API is dissolved into thefirst solution.

Next, a second solution, which is aqueous, is obtained that includes amucoadhesive hydrophilic polymer and a buffer. The mucoadhesivehydrophilic polymer can be sodium hyaluronate, xanthan gum, guar gum,carboxymethylcellulose, 1-4 beta glucan, poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), tamarind seedpolysaccharide, sodium alginate, polycarbopol, and polycarbophil, ormixtures thereof. The buffer can be, but is not limited to, sodiumacetate, sodium dihydrogen phosphate, disodium hydrogen phosphate,potassium dihydrogen phosphate, dipotassium hydrogen phosphate,ε-aminocaproic acid; amino acid salts such as sodium glutamate, boricacid, and citric acid. Preferably, the buffer is a phosphate buffer. Thesecond solution can be maintained at a temperature of 5-55° C.,preferably 40-55° C.

The first solution and the second solution are mixed together to form acombined nano/micro-dispersion. The weight ratio between the first andsecond solution can be 1:1 to 1:15 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:4, 1:15). Preferably, the ratiois 1:9.

As mentioned above, the mixing of the first and second solutions to forma combined nano/micro-dispersion is accomplished by a high energy mixingprocess. The high energy mixing process can be, e.g., high shear mixing,sonication, or a combination of both processes.

The combined nano/micro-dispersion is subjected to microfluidization toform a nano-dispersion. The microfluidization can be performed, e.g., byhigh pressure homogenization. The microfluidization can be carried outat 40-55° C. for a period of 8-24 h.

The nano-dispersion thus formed can be incubated at 2-5° C. for a periodof 12-24 h to form a liquid crystalline drug delivery system.

An alternative embodiment of the method includes, in addition to all ofthe aforementioned steps, a step of mixing the nano-dispersion for 16-24h prior to incubating them at 2-5° C. for a period of 12-24 h. Thismixing step can be accomplished by high shear mixing. Additionally, thismixing step can be performed at −10 to 15° C. In one embodiment, themixing is performed at −10 to −1° C. In an alternative embodiment, themixing is performed at 8-15° C. In another embodiment, the mixing isperformed at room temperature.

The first solution mentioned above can also be formed by adding, inaddition to the lipidic component and the alcohol, PEG-400, PPG,PEG-stearate, poloxamer 407, tyloxapol, polysorbate 80, and castor oil.In another embodiment, cholesterol is also added to form the firstsolution. In an additional embodiment, PLGA is added to the firstsolution.

In a particular embodiment of the method, the first solution is formedfrom, by weight, 10% phosphatidylcholine and medium chain triglycerides,10% cetyl alcohol, 20% PEG-400, 10% PPG, 7% PEG-stearate, 2.2% poloxamer407, 1% tyloxapol, 1% polysorbate 80, and 38% castor oil. The firstsolution can also be formed with, in addition to these components, 2% byweight cholesterol. Fluticasone propionate is dissolved into the firstsolution at 1%. In this particular embodiment, the second solutioncontains, by weight, 0.2% sodium phosphate monobasic, 0.5% sodiumphosphate dibasic, and 1.5% sodium hyaluronate. The first and secondsolutions are mixed at a 1:9 weight ratio.

The first solution described above can be produced in a step-wisefashion. For example, an API can first be solubilized in the lipidiccomponent and the alcohol. This solubilization can be performed at atemperature required to dissolve the desired amount of API. Thetemperature can be, e.g., 25-65° C. After solubilizing the API, one ormore of the additional excipients described above can be added to formthe first solution. The temperature of the first solution can be loweredto 30-45° C. if a higher temperature was used to solubilize the API.

As described in detail, supra, the first solution is mixed, by a highenregy mixing process, with the second solution containing amucoadhesive hydrophilic polymer. In an alternative embodiment, themucoadhesive hydrophilic polymer is omitted from the second solution andadded after the microfluidization step but before the final incubationstep. This alternative process may be necessary when using specificmucoadhesive hydrophilic polymers which are sensitive to shear forces inthe mixing steps.

Also mentioned above is a method for treating an ocular disorder in asubject. The method includes the steps of identifying a subject havingan ocular disorder and administering to an eye of the subject the liquidcrystalline drug delivery system described above.

A skilled person in the art can identify the subject having an oculardisorder by routine methods in the art, e.g., an eye exam. The oculardisorders that can be treated with the liquid crystalline drug deliverysystem include but are not limited to post-operative inflammation,inflammation, allergic rhinitis, allergic conjunctivitis, meibomiangland dysfunction, infection, conjunctivitis, keratitis, ulcers,blepharitis, glaucoma, uveitis, diabetic macular edema, diabeticretinopathy, age-related macular degeneration, endophthalmitis,choroidal neovascularization, tear duct dysfunction, corneal blebs, anddry eye disease.

The liquid crystalline drug delivery system can be administered to theeye by eye-drops, spray, and injection. In a particular embodiment, theliquid crystalline drug delivery system is administered by vitreousinjection.

The liquid crystalline drug delivery system to be administered ispreferably formulated with an API known to be effective for treating theocular disorder. The API can be, but is not limited to fluticasonepropionate, dexamethasone, betamethasone, budesonide, triamcinoloneacetonide, methyl prednisolone, cortisone, beclometasone, fluticasonefuroate, deoxycorticosterone acetate, loteprednol etabonate,difluprednate, fluorometholone, rimexolone, travoprost, moxifloxacin,prednisolone acetate, posaconazole, budesonide, netilmycin, ormupirocin.

In other embodiments, the liquid crystalline drug delivery system can beformulated with non-steroidal anti-inflammatory drugs, including but notlimited to nepafenac, bromfenac sodium, diclofenac, flurbiprofen sodium,ketorolac tromethamine, and flurbiprofen sodium.

In another embodiment, anti-microbials can be incorporated into theliquid crystalline drug delivery system. The anti-microbials include,but are not limited to, tobramycin, netilmycin, erythromycin,bacitracin, azithromycin, ciprofloxacin, gatifloxacin, gentamycinsulfate, levofloxacin, moxifloxacin hydrochloride, ofloxacin,sulfacetamide sodium, Polymyxin B sulfate, sulfacetamide, neomycinsulfate, bacitracin zinc, and gramicidin.

The liquid crystalline drug delivery system decribed above can beformulated with a hydrophobic drug. Examples of hydrophobic drugsinclude, but are not limited to, ROCK inhibitors, EGFR inhibitors, A-1agonists, PARP inhibitors, SOD mimetics, PPAR agonists, WNT inhibitors,SYK-specific inhibitors, JAK-specific inhibitors, SYK/JAK orMulti-Kinase inhibitors, MTORs, STAT3 inhibitors, VEGFR/PDGFRinhibitors, c-Met inhibitors, ALK inhibitors, mTOR inhibitors, PI3Kδinhibitors, PI3K/mTOR inhibitors, p38/MAPK inhibitors, macrolides, azolederivatives, prostaglandins, NO-releasing agents, peptides, NSAIDs,steroids, antibiotics, antivirals, antifungals, antiparsitic agents,blood pressure lowering agents, cancer drugs or anti-neoplastic agents,immunomodulatory drugs, diagnostic agents, and anti-oxidants.

Combinations of any of the above-mentioned APIs can also be incorporatedinto the liquid crystalline drug delivery system.

Alternatively, the liquid crystalline drug delivery system can beformulated without an API and administered for treating ocularconditions in which tear production is impaired or absent.

Without further elaboration, it is believed that one skilled in the artcan, based on the description above, utilize the present invention toits fullest extent. The specific examples below are to be construed asmerely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever.

EXAMPLES Example 1 Optimization of Process Development

As mentioned above, the nanoparticles (Phase I) are the API-containingportion of the formulation. The API is dissolved in a mixture ofmembrane-mimetic excipients that are co-soluble and also solubilize theAPI. The excipients used should be generally regarded as safe (GRAS) andUS FDA-approved for ophthalmic use. A high solubility of API in Phase Iwill ensure a high concentration of the API in the finalnano-dispersion. Phase I, containing at least one hydrophobic and onehydrophilic component, is also the dispersed phase in the liquidcrystalline dispersion system.

Solubility of APIs were examined in different Phase I mixtures.

Initially, the solubility of fluticasone propionate was determined in 10g of Phase I composed of a 7:3 (w/w) mixture of PHOSAL™ Medium ChainTriglycerides (“MCT”) and polyethylene glycol-400 (PEG-400). MCT, amixture of phosphatidylcholine and medium chain triglycerides, is thehydrophobic component and PEG-400 is the hydrophilic component in thisexemplary Phase I. MCT and PEG-400 were added into a glass vial and themixture was vortexed for 2 min until the components formed a homogeneousmixture. Fluticasone propionate was added in an incremental manner andstirred at 850 rpm using a Scilogex MSH 280 Pro hot stir plate at atemperature of 37° C. Stirring was continued until saturation solubilitywas reached. Fluticasone propionate had a saturation solubility of 108.7mg in 10 g of this Phase I mixture.

In order to increase the solubility of the fluticasone propionate inPhase I, mineral oil (Drakeol 600LT) was added into the composition. APhase I containing MCT: PEG-400: mineral oil weight ratio of 7:2:1 wasprepared as described above. The total amount of Phase I was 10 g.Fluticasone propionate was added in an incremental manner and themixture was stirred for 5 min. and vortexed for 5 min after eachaddition of fluticasone propionate into the mixture. In this Phase I,98.2 mg of fluticasone propionate dissolved completely in 37 min.

Another Phase I mixture containing PEG-400, polypropylene glycol (PPG),and cremophor in a weight ratio 2.9:7:0.1 was also evaluated. Cremophorwas used because it is widely used as a co-solvent to enhance thesolubility of poorly soluble drugs. The saturation solubility of thefluticasone propionate in this Phase I was 103.7 mg in 10 g afterstirring at 990 rpm at 45° C. for 20 min.

The solubility of fluticasone propionate was also evaluated in 5% cetylalcohol in ethanol. In this Phase I, 200.30 mg of fluticasone propionatewas completely dissolved in 10 g when stirred at 1200 rpm for 2 min at45° C.

Another Phase I mixture was produced containing 30% w/w PEG-400, 60% w/wPPG, 5% w/w MCT, 0.25% w/w cetyl alcohol, and 4.75% w/w ethanol. In 10 gof this Phase I, 180 mg fluticasone propionate dissolved completelyafter stirring at 1280 rpm for 15 min at 45° C.

A different API, namely, triamcinolone acetonide was also tested withthis Phase I. It was found that 220 mg of triamcinolone acetonidedissolved in 10 g of Phase I after stirring at 1280 rpm for 20 min at45° C. Triamcinolone acetonide is a non-steroidal anti-inflammatory drughaving low aqueous solubility.

Further modifications of Phase I were tested with the aim of enhancingAPI solubility further. To achieve this aim, additional hydrophobiccomponents were introduced into Phase I.

A Phase I containing 30% w/w PEG-400, 20% w/w PPG, 0.5% w/w cetylalcohol, 9.5% w/w ethanol, 5% w/w MCT and 35% w/w Castor oil wasproduced. Triamcinolone acetonide was added incrementally and themixture stirred at 840 rpm using a Scilogex MSH 280 Pro set to 45° C.The saturation solubility for triamcinolone acetonide in 10 g of thisPhase I was 230 mg.

Phase II is the aqueous phase, into which Phase I is poured, pumped, orinjected, and subsequently mixed. Phase II, an aqueous solutioncontaining only hydrophilic excipients, provides a buffer whichmaintains the pH and protects the API from pH-related instability in thefinal formulation. The optimization of Phase II was monitored by mixingit with Phase I to form Phase III. As mentioned above, Phase III is theterm used for the emulsion that is formed when Phase I and Phase II aremixed using high energy processes.

A Phase II was formulated containing 0.5% w/w sodium hyaluronate, 0.63%w/w sodium chloride, 0.3% w/w sodium phosphate dibasic, 0.04% w/w sodiumphosphate monobasic, and 98.56% w/w distilled H₂O (dH₂O) at pH 6.5±0.1.

One part by weight of a Phase I containing 30% w/w PEG-400, 20% w/w PPG,0.5% w/w cetyl alcohol, 9.5% w/w ethanol, 5% w/w MCT, and 35% w/w Castoroil was injected into 9 parts by weight of the Phase II at a flow rateof 0.5 g/min. to form Phase III. The mixing process was carried outusing a Heilscher UP200S ultrasonic processor coupled with a sonotrodeS3 microtip, with the processor set to 25% amplitude and 0.5 cycle. ThePhase II was contained within a jacketed vessel set to 32° C.

The mean particle size of this Phase III, as measured by a Horiba LA952particle analyzer, was found to be 26 μm. The emulsion produced by thisprocess was not uniform in particle size; the variance for particle sizedistribution was 2157.60.

In an attempt to reduce the non-uniformity and large particle size ofPhase III, polyethylene glycol stearates (PEG-stearates) were added toPhase I. PEG—stearates are mixtures of distearate esters of mixedmacrogols (polyoxyethylene polymers) and corresponding free glycols.They are typically used as emulsifying and solubilizing agents inpharmaceutical compounds. Additionally, to achieve higher levels ofhydrophobic content and to avoid high levels of ethanol in theformulation, a greater amount of MCT was used, and cetyl alcohol wasused as described above. In order to avoid the clumping of cetyl alcoholat lower temperatures, the hydrophobic components were mixed at 67° C.at 250 rpm. A Phase I was produced that contains 28% w/w PEG-400, 10%w/w PPG, 10% w/w cetyl alcohol, 10% w/w Phosal MCT, 2% w/w PEG-stearateand 40% w/w Castor oil. This Phase I (1 part by weight) was injectedinto 5 parts by weight Phase II (0.5% w/w sodium hyaluronate, 0.63% w/wsodium chloride, 0.3% w/w sodium phosphate dibasic, 0.04% w/w sodiumphosphate monobasic, 98.56% w/w dH₂O) at a flow rate of 0.5 g/min toform a Phase III. The mixing process was carried out as described abovewith the ultrasonic processor set to 25% amplitude and 0.5 cycle. ThePhase II was housed in a jacketed vessel set to 25° C. The mean particlesize of the Phase III was found to be 30 μm. The emulsion produced bythis process was better in uniformity of particle size, the variance forparticle size distribution being 653.4, but the particle size hadincreased.

To further reduce the particle size, the ratio between Phase I and PhaseII was varied while keeping their compositions constant. To form a PhaseIII, one part by weight of Phase I and 15 parts by weight of Phase II(both described in the previous paragraph) were mixed using theultrasonic processor set to 30% amplitude. and 1 cycle. The Phase II washoused in a jacketed vessel set to 25° C. The emulsion produced by thismodified process was better in uniformity of particle size, with avariance for particle size distribution of 6.35 and a mean particle sizeof 3.6 μm. In this modification, the amplitude setting on the ultrasonicprocessor was 30%, as opposed to 25% described above. Despite theincreased amplitude employed, the temperature of the final Phase IIIafter sonication was found to be lower than the Phase III emulsionshaving a 1:5 ratio between Phase I and Phase II.

Thus, two factors were found to affect the particle size in a favorablemanner, a lower Phase III temperature and a lower Phase I to Phase IIratio.

Lowering the ratio of Phase I to Phase II from 1:5 to 1:15 would havethe effect of diluting the concentration of an API in the finalformulation. Therefore, additional Phase I/II ratios were tested thatwould not lower the drug concentration as much as the 1:15 ratio.

The composition of Phase I and Phase II mentioned above were keptunchanged A Phase III was formed from a 1:11 ratio of Phase I to PhaseII. Again, the mixing process was carried out using the ultrasonicprocessor set to 30% amplitude and 1 cycle. The Phase II was housed in ajacketed vessel set to 25° C. The mean particle size of the Phase IIIthus produced was 5.87 μm, with a variance of particle size distributionof 86.30. The ratio between Phase I and Phase II played an importantrole in the size of particles in the emulsion. A ratio that provides anoptimal concentration of the drug (without diluting it too much) is ofparamount importance in forming an efficient drug delivery system.

As mentioned above, keeping the temperature of Phase III low was animportant factor to yield smaller particle sizes. Additionally,increasing the sonication duration was another factor. Increasing theduration of sonication is problematic as this leads to an increase inPhase III temperature. This increase, in turn, contributes to theincreased particle size. To avoid an increase in the particle size dueto a rise in temperature, bath sonication of the Phase III wasintroduced into the process.

The composition of Phase I and Phase II were kept unchanged and thephase ratio was kept at 1:11. Phase II was housed in a jacketed vesselset to 25° C. The ultrasonic mixing process was carried out as describedabove using a 30% amplitude and 1 cycle to form Phase III. The Phase IIIthus formed was subjected to 15 min. of bath sonication. The meanparticle size of the Phase III was 5.48 μm with a variance of particlesize distribution of 8.23. The temperature of Phase III was 28° C.,indicating that the Phase III temperature remained relatively constant.The addition of a bath sonication process in the formulation developmentwas a favorable change, as the particle size remained the same, whilethere was a reduction in the variance from 86.30 to 8.3, pointing to anincrease in uniformity. In the case of liquid crystalline nanoemulsions,it has been observed that the presence of larger particles, as evidencedby a large variance, in the emulsion leads to coalescence and eventuallyphase separation in the emulsion. As such, the variance is also anindicator of stability.

Various surfactants were tested in an attempt to further reduce particlesize. Tyloxapol, a nonionic liquid polymer of the alkyl aryl polyetheralcohol type and a surfactant used to aid liquefaction, was added aboveits critical micelle concentration (CMC) of 0.018 mM to Phase II.

The composition of Phase I was kept unchanged (28% w/w PEG-400, 10% w/wPPG, 10% w/w cetyl alcohol, 10% w/w Phosal MCT, 2% w/w PEG-stearate, 40%w/w Castor oil) and Phase II was formulated with 0.5% w/w sodiumhyaluronate, 0.63% w/w sodium chloride, 0.3% w/w sodium phosphatedibasic, 0.04% w/w sodium phosphate monobasic, 4% 0.1 mM tyloxapolsolution, and 98.56% w/w dH₂O. Phase II was housed in a jacketed vesselset to 25° C. A Phase III was formed from a 1:11 Phase Ito Phase IIratio by first mixing ultrasonically as described above with theprocessor set to 30% amplitude and 1 cycle. The Phase III mixture wassubjected to 15 mM. of bath sonication. The mean particle size of thePhase III thus formed was 1.81 μm, with a variance of particle sizedistribution of 1.02, indicating that the emulsion had a uniformparticle size. The temperature of the Phase III was consistently 29° C.,indicating that the temperature remained nearly unchanged. The additionof tyloxapol in the formulation development was favorable, as theparticle size decreased by 600%, and there was a reduction in thevariance from 8.3 to 1.02, indicating a stable emulsion.

Another surfactant that was tested was polysorbate 80. Polysorbate 80was used below its CMC. The composition of Phase I was kept unchanged,while Phase II was formulated with 0.5% w/w sodium hyaluronate, 0.63%w/w sodium chloride, 0.3% w/w sodium phosphate dibasic, 0.04% w/w sodiumphosphate monobasic, 0.00015% w/w polysorbate 80, and 98.56% w/w dH₂O.The Phase II was housed in a jacketed vessel set to 25° C. The ratio ofPhase I to Phase II was kept at 1:11. The mixing process was carried outas described in the preceding paragraph. After the formation of PhaseIII, it was subjected to 15 min. of bath sonication. The mean particlesize of Phase III was 53.75 μm, with a variance of particle sizedistribution of 6.05, indicating that the Tween addition below its CMCdid not reduce the particle size.

A similar study was conducted to evaluate the effect on particle size oftyloxapol below its CMC. The presence of tyloxapol did reduce theparticle size as compared to the Phase III emulsion produced withpolysorbate 80 below its CMC. Moreover, this Phase III showed gooduniformity in particle size, indicating a stable formulation.

Experiments were conducted to evaluate a synergistic effect oftemperature and tyloxapol. These experiments were conducted at lowertemperature, which was achieved by setting the Phase II jacketed vesselto 15° C. The resultant Phase III temperature after sonication was 22°C. Although a lower Phase III temperature was achieved, the size of theparticles was 7.14 μm with a variance of 136.18, both values larger thanthose obtained at higher temperatures. The increase in particle size wasattributed to the presence of sodium hyaluronate, which increases theviscosity of the Phase II at lower temperature. Thus, the presence of astabilizer was eclipsed by the temperature of the process, and thetemperature of the process proved to be a more important factor for theemulsion.

Another set of studies were conducted to evaluate the effect ofsonication intensity on the particle size. It was observed that when themixing process was carried out as above using the ultrasonic processorset to 40% amplitude (as opposed to 30% described above) and cycle 1,the mean particle size was reduced to 2.41 μm with a variance of 1.58.It was evident that higher sonication leads to smaller particle size.Additionally, it was observed from a series of experiments thatsonication intensity played a more important role than the temperatureof the Phase III mixing phase in determining particle size.

Since higher ultrasonic amplitude was found to be a favorable factor,the ultrasonic intensity was raised to 60% amplitude at cycle 1. Thephase ratio was reduced to 1:10 (Phase I to Phase II), instead of 1:11,and the Phase II was housed in a jacketed vessel set to 25° C. After theformation of Phase III, it was subjected to 15 min of bath sonication.The mean particle size was found to be 1.09 μm, and the emulsion wasquite uniform with a variance of only 0.2116.

A series of experiments were carried out to further evaluate the effectof sodium hyaluronate, i.e., the sodium salt of hyaluronic acid, whichis a glycosaminoglycan found in various connective, epithelial, andneural tissues. Sodium hyaluronate, a long-chain polymer containingrepeating disaccharide units of sodium-glucuronate-N-acetylglucosamine,occurs naturally on the corneal endothelium. Sodium hyaluronate wasintroduced into Phase II for two reasons: (a) it functions as a tissuelubricant and facilitates wound healing and (b) it increases theviscosity of the formulation, thereby increasing the contact timebetween the delivery system and the target organ. The composition ofPhase I was kept unchanged (28% w/w PEG-400, 10% w/w PPG, 10% w/w cetylalcohol, 0% w/w MCT, 2% w/w PEG-stearate, 40% w/w Castor oil). Variousconcentrations of sodium hyaluronate were added to Phase II (0.63% w/wsodium chloride, 0.3% w/w sodium phosphate dibasic, and 0.04% w/w sodiumphosphate monobasic, dH₂O). The results are shown in Table 1 below. Itwas observed that adding 0.4% w/w sodium hyaluronate to Phase IIresulted in emulsions with lower particle sizes (50% of particles below1 μm, i.e., D50<1 μm), while other concentrations of sodium hyaluronateresulted in particle sizes above 1 μm.

TABLE 1 Particle size distribution as a function of sodium hyaluronateconcentration. Values shown are the particle size distributions (μm). HA(%) D50 (μm) D90 (μm) mode (μm) 0.1 1.02 1.78 1.09 0.2 1.18 1.72 1.200.3 2.3 4.39 2.41 0.4 0.9 1.49 0.9 0.5 1.02 1.78 1.09 D50 = 50% of theparticles are smaller than the value shown; D90 = 90% of the particlesare smaller than the value shown; mode = statistical mode.

To further reduce the size of the particles in the emulsion, high shearmixing was introduced to the process. A Silverson LSMA mixer fitted witha high shear screen was used to mix Phase I and Phase II.

In a specific example, the composition of Phase I was 27% w/w PEG-400,10% w/w PPG, 10% w/w cetyl alcohol, 10% w/w MCT, 2% w/w PEG-stearate,40% w/w castor oil, and 1% w/w dexamethasone. The method of preparationof Phase I was modified from the above in order to incorporate thedexamethasone. The hydrophobic components were weighed and mixed at50-55° C. to form a homogenous mixture. The hydrophilic components werethen added and the mixture was continuously stirred to homogeneity.Finally, the dexamethasone was added and the mixing continued at 50-55°C. until the drug dissolved completely, resulting in a clear solution.

Phase II contained 0.4% w/w sodium hyaluronate, 0.63% w/w sodiumchloride, 0.3% w/w sodium phosphate dibasic, 0.04% w/w sodium phosphatemonobasic, 0.28% w/w PEG-stearate, 0.14% w/w polysorbate 80, and 98.21%w/w dH₂O.

To form 550 g of Phase III with a Phase I/Phase II ratio of 1/10, 500 gof Phase II was poured into a jacketed vessel set at 25° C. Phase I (50g) at 39° C. was injected into Phase II at 0.5 g/min. (±0.1), whilebeing mixed with a high shear screen at 10,000 rpm for 15 min The mixerwas reduced to 6000 rpm for the next 60 min. The mean particle size ofPhase III thus formed was 0.85 μm (±0.44) with a variance of 0.196.

To improve the formulation further, the compositions of Phase I andPhase II described in the preceding paragraph were modified by raisingthe concentration of PEG-stearate in Phase I to 3% and omitting it fromPhase II. Additionally, tyloxapol was added to Phase II at 0.3% w/w.

To produce 100 g of Phase III with a Phase I/Phase II ratio of 1/10, 90g of Phase II was cooled prior to the mixing step to reduce the overalltemperature of the process, after which it was poured into a jacketedvessel set at 15° C. Phase I (10 g) at 50° C. was injected into Phase IIat 0.5 g/min (±0.1), while being mixed by Silverson LSMA mounted withemulsor screen at 6,000 rpm for 60 min. and 10,000 rpm for the next 15min. at 30° C. The mean particle size of Phase III was 4.58 μm (±9.58)with a large variance of 91.58. Since very large particles were present,the head of the mixer was changed from an emulsor screen to a squareholed high shear screen, as the latter provided more shear. The emulsionwas further mixed for an additional 5 min. Mixing with the high shearscreen reduced the mean size of the particles to 0.68 μm (±0.38)

The osmolality of the formulations is an important factor in theformulation development. When measured, the osmolality of theformulation described in the preceding paragraph was found to be 360mOsm/kg. To reduce the osmolality of the formulation, the amount ofPEG-400 was reduced to 20% w/w and the amount of PEG-stearate wasincreased to 7% w/w. Moreover, sodium hyaluronate was removed, as itincreased the viscosity of Phase II, thereby contributing to largerparticle size during mixing.

A 100 g amount of Phase III with a Phase I/Phase II ratio of 1/10 wasformed by mixing 500 g of Phase II in a jacketed vessel set at 25° C.with 50 g of Phase I at 50-55° C. The Phase I was injected into Phase IIat 1.0 g/min (±0.1) while being mixed with a high shear screen at 700rpm for 15 min. at 25° C., 6000 rpm for 60 min, at 19° C., followed by10,000 rpm for 10 min at 30° C. The mean particle size of Phase III was0.141 μm (±13.38) with a large variance of 184.14.

The flow rate of Phase I into Phase II is an important factor thataffects the particle size of the Phase III formulation. This was moreevident in the formulation development process that employed high shearmixing. A series of experiments was carried out to evaluate the effectof Phase I flow rate on the particle size of the Phase III emulsion.Variance was considered as a measure of stability instead of mean, mode,or median size, as variance is an indicator of the overall uniformity ofthe emulsion. For example, if the emulsion has a mean particle size of0.1 μm and a variance of 2.0 μm, it indicates that the emulsion has asmall population of particles that are larger than desired and theirpresence may eventually lead to coalescence and phase separation. Theresults are shown in Table 2 below. It is evident that increased flowrate led to a higher variance,.i.e., low stability.

TABLE 2 Variance as a function of flow rate Flow rate (g/min.) variance0.475 0.75 1.04 2.46 2.07 9.54 2.08 22.75 6.57 197.96

There was a considerable difference in particle size betweenformulations prepared with various mixing processes. As described above,three different mixing protocols were used in the development of theformulations. Mixing Phase I and Phase II by high shear mixing resultedin the percentage of particles below 200 nm consistently higher than80%. Mixing Phase I and Phase II by sonication alone producedformulations having less than 0.8% of particles of 200 nm or less, whilea combination of sonication and mixing resulted in formulations having12-50% of particles less than 200 nm in size.

Various APIs were used to test the feasibility of this drug deliveryplatform. It was found that the platform was effective for producingnanodispersions using a number of poorly aqueous soluble non-steroidalanti-inflammatory drugs. Exemplary drugs are as follows, presented withtheir corresponding D50 and D90 values: dexamethasone (D50=0.139 μm;D90=0.170 μm), triamcinolone acetonide (D50=0.134 μm; D90=0.197 μm), andfluticasone propionate (D50=0.141 μm; D90=0.181 μm).

Example 2 Analysis Methods

Imaging of a 20 μL droplet of the dispersions was performed by placingit on a microscope slide and covering it with a glass coverslip, takingcare to maintain the integrity of the emulsion. The dispersions wasexamined under crossed polarizers using a 100× objective of an OlympusBX51P Polarizing Light Microscope, under an oil-drop. Bothdrug-containing and drug-free dispersions were examined

Particle sizing was carried out by adding approximately 20 μL of thePhase III dispersion in a solution of 2% w/w glycerin, 0.1% w/w sodiumpyrophosphate decahydrate. Particle size distribution of the Phase IIInano-dispersions were measured using a Horiba LA-950V2 particle sizeanalyzer at room temperature, i.e., 22-25° C.

Encapsulation efficiency (mg/G) was measured by placing 1.0 g ofdrug-containing Phase III into a 1.5 mL centrifuge tube and centrifugingit at 6000 rpm for 10 minutes using an Eppendorf Centrifuge 5145D atroom temperature. 100 μL of the centrifugate was transferred to an HPLCvial containing 900 μL of 75% acetonitrile/25% water. The samples weremeasured for concentration of drug, e.g., dexamethasone, by HPLC atλ_(max)=239 nm. The concentration was calculated as mg drug per g of thePhase III dispersion.

In-vitro drug release was determined at 37° C., pH 7.4 as follows: ThePhase III dispersion (1 g) was transferred into a Spectra/PoreFloat-A-Lyzer G2 dialysis device, which was then placed into a 50 mLlocking centrifuge tube containing 40 g of 1%hydroxylpropyl-β-cyclodextrin (HP-β-CD) in phosphate buffer at pH 7.4,37° C. The entire assembly was loaded onto a Robbins Scientific Model400 rotating incubator. At each time point, 1 mL of sample was retrievedand fresh buffer added to replace the volume removed. Samples weremeasured for drug content using HPLC at λ_(max)=239 nm.

Ex-vivo corneal permeability testing is a useful tool to screenformulations for their ability to penetrate ocular tissues. Usingfreshly excised cornea, formulations can be tested for their ability todiffuse across the cornea membrane. The ability to diffuse throughbiological membranes is directly related to the formulation excipients,its physical state (e.g., suspension, solution, emulsion, dispersion)and its partition coefficient (P) and log P.

Fresh calf eyes were obtained from a nearby slaughterhouse and thecorneas carefully excised using sterile technique. The corneas must befreshly excised and used within 1-2 hours. The corneas were excised in asterilized laminar flow hood, in a Class 100 environment. The excisionswere performed by first draining the aqueous humor followed by carefullycutting out the cornea using a scalpel for the initial incision; forcepsand scissors were used to cut the remaining tissue. The excised corneawas stored in a petri dish with a small amount of a hydrating solutioncontaining, by weight, 0.1% glutathione, 0.051% disodium phosphate, and99.45% H₂O at a pH of 7.0.

The solubility of the API in the receptor fluid is very critical incorneal permeability experiments. The saturated solubility of the drugin the receptor fluid must be much greater than the total theoreticalconcentration of the drug in the receptor solution. The composition of atypical receptor fluid is, by weight, 1% HP-β-CD, 0.051% disodiumphosphate, 0.017% sodium phosphate monobasic, and 98.55% H₂O.

Corneal permeability studies were performed using a Franz-Cell diffusionchamber system. The Franz-Cell system consists of 6 in-line jacketedcells mounted on a single unit with individual magnetic stir plates,with each cell connected to the main system water jacket. The jacket wasmaintained at 37° C. for the duration of the experiment using arecirculating heating bath. Each cell consists of a donor cell on thetop where a known volume of the formulation is pipetted and a receptorcell with a sampling side arm below. The joint between the donor andreceptor cell is upward-convex, mimicking the shape of the cornea. Eachreceptor cell holds 5 mL of receptor fluid, and each donor cell holds200 μL of the formulation being studied.

The receptor fluid was added to each receptor cell using a syringeequipped with a needle. The solution was slowly added, until there was aconvex meniscus on the donor cell joint. The volume was recorded, andthe remaining cells filled.

After weighing the cornea, it was placed on top of the receptor-donorcell joint using a pair of forceps, with care taken to ensure that therewere no folds in the cornea and no bubbles. Once in place, donor cellcaps were attached carefully, and locked in place with a metal clip.

Samples were added in rapid succession by depositing 200 μL of theformulation into each donor chamber using a calibrated pipet and thetimes recorded. The donor chamber and sampling arm were sealed to ensureno significant evaporation has occurred.

Samples were withdrawn from the receptor cells at 2, 4, 6, 7, and 22hours. The samples were analyzed for API content by HPLC as describedabove.

Flux (J) is the amount of drug crossing the membrane per unit time. Itis given in units of mass/area/time. Flux can be calculated by theformula: J=Q/(A·t), where Q is the quantity of compound traversing themembrane in time t, and A is the area of exposed membrane in cm². Theunits for flux are weight (micrograms)/cm²/min.

Example 3 Preparation of Nanostructured Dispersions Containing 0.1%Fluticasone Propionate

Preparation of Phase I

A 30 g amount of Phase I was prepared by adding 3 g of PHOSAL® MixedChain Triglycerides (“MCT”), 3 g of cetyl alcohol, and 11 g of castoroil into a pre-tared glass beaker. The mixture was heated to 55° C. withcontinuous stirring on a hot plate to form a homogenous mixture. To thismixture, 6 g of polyethylene glycol-400, 3 g of polypropylene glycol,2.1 g of PEG-40-stearate, 0.666 g of Poloxamer 407, 0.3 g of Tyloxapoland 0.3 g of Tween 80 were added, followed by stirring at 55° C. Afterensuring a homogenous mixture was achieved, the heat component of thestir plate was switched off. Fluticasone propionate (0.24 g) was addedto the mixture once it cooled to 40° C.-45° C. The mixture was stirreduntil the fluticasone propionate was completely solubilized into ahomogenous, clear solution with no visible particulates, thereby formingPhase I.

Preparation of Phase II

A 300 g amount of Phase II was prepared from 0.051 g of sodium phosphatemonobasic, 0.156 g of sodium phosphate dibasic, and 299.796 g ofdistilled H₂O (dH₂O) were added into a tared glass beaker. This mixturewas stirred until the sodium salts were completely dissolved, therebyforming Phase II. The pH of Phase II was 7.3.

Preparation of Phase III

Phase III is the term used for the dispersion that is formed when PhaseI and Phase II are mixed using high energy processes. In this example,high shear mixing was used to obtain the final dispersion.

To obtain 100 g of Phase III, 10 g of Phase I was injected into 90 g ofPhase II at a flow rate of 1 g/min and the mixture continuously mixedusing a Silverson LSMA high shear laboratory mixer. During theinjection, Phase I was maintained at 40-45° C. and Phase II was cooledto 8° C. More specifically, Phase II was poured into a jacketed vesselconnected to a chiller set at −10° C.

Two mixing speeds were used to obtain the final dispersion, i.e., PhaseIII. A high speed of 7,500 RPM was used when Phase I was being injectedinto Phase II. Upon complete addition of Phase I, the mixing speed wasreduced to 5,040 RPM for the remainder of the mixing period. The mixingwas carried out for a total of 150 minutes. In this example, the highmixing rate was used for the first ten minutes, and then the lower ratewas used. The final concentration of fluticasone propionate was 0.1% byweight of Phase III.

The statistical mode of particle size in the dispersion was 120 nm. Themiddle of the dispersion was 122 nm, and 85% of all the particles weresmaller than 300 nm. The dispersion appeared milky white, homogeneous,and stable upon storage at room temperature.

When examined by polarized optical microscopy, the dispersion displayeda unique nanostructure, akin to a liquid crystalline state. Thedispersed phase is semi-solid, rendered so by the intercalation of PhaseII into Phase I. The nano-size of the dispersion renders it suitable forpermeation into tissues.

Example 4 Preparation of Nanostructured Dispersions ContainingDexamethasone

Preparation of Phase I

A 30 g amount of Phase I was prepared by mixing 3 g of MCT, 3 g of cetylalcohol, and 12 g of castor oil in a glass beaker. This mixture washeated to 55° C. with continuous stirring on a hot plate to form ahomogenous mixture To this mixture, 6 g of polyethylene glycol-400, 3 gof polypropylene glycol, 2.1 g of PEG-40-stearate, and 0.6 g ofPoloxamer 407 were added, followed by continued stirring at 55° C. Aftera homogenous mixture was formed, the heat component of the stir platewas switched off. Dexamethasone (0.3 g) was added to the mixture oncethe mixture cooled to 40-45° C. The mixture was stirred until thedexamethasone was completely solubilized.

Preparation of Phase II

A 300 g amount of Phase II was prepared by mixing 0.051 g of sodiumphosphate monobasic, 0.156 g of sodium phosphate dibasic, and 299.796 gof dH₂O in a glass beaker. This mixture was stirred until the sodiumsalts were completely dissolved. The pH of Phase II was 7.3.

Preparation of Phase III

Phase III was formed as follows: 90 g of Phase II was cooled to 8° C. bypouring it into a jacketed vessel connected to a chiller set at −10° C.A 10 g amount of Phase I, maintained at 40-45° C.; was injected intoPhase II at a flow rate of 1 g/min with continuous mixing using aSilverson LSMA high shear mixer.

Like Example 3 described above, two mixing speeds were used to obtainthe final dispersion, i.e., Phase III. Initially, a high speed of 10,000RPM, i.e., primary mixing, was used while Phase I was being injectedinto Phase II. After all of the Phase I was introduced, the mixing wascarried out at a lower rate of 5,040 RPM, i.e., secondary mixing, forthe remainder of the mixing period. The mixing was carried out for atotal of 150 min, where the high mixing rate was used for the first tenminutes followed by the lower rate. The final concentration ofdexamethasone was 0.1% by weight of Phase III.

Analysis of the dispersion revealed that the median (d50) of theparticle size distribution was 143 nm, the mode was 141 nm, and 90% ofthe dispersed nano-structured particles were smaller than 245 nm. Thenanostructured dispersion was stable over time at room temperature. Whenexamined microscopically, the dispersion demonstrated an orderedmicrostructure, indicative of an ordered, but liquid-like state.

Additional analysis revealed that the dexamethasone was encapsulated at0.845 mg/G. An in-vitro drug release assay indicated that at least 25%of the dexamethasone was released over 3 hours, indicating a highlybioavailable formulation.

The corneal permeability of the nanostructured formulation of 0.1%dexamethasone was tested as described above. Approximately 35% of thedexamethasone applied to the corneas was released into the receptorsolution over a 22 hour period, indicating a high bioavailability of theformulation. In contrast, a suspension of dexamethasone tested in thesame assay displayed a fairly low corneal permeability of <5% in 22hours. Additionally, after the diffusion study was complete, the corneaswere extracted in acetonitrile and analyzed for dexamethasone content. Asubstantial depot-like effect was observed in the corneas treated withthe formulation, indicating that a sustained release effect can beachieved with this formulation. More specifically, the amount ofdexamethasone extracted from the corneas averaged 35% of the totalinitially loaded onto the corneas.

Example 5 Preparation of Nanostructured Dispersions ContainingLoteprednol Etabonate

Preparation of Phase I

A 30 g amount of Phase I was prepared as described above in EXAMPLE 4,except that loteprednol etabonate (0.3 g) was added instead ofdexamethasone.

Preparation of Phase II

A 300 g amount of Phase II was prepared also as described in EXAMPLE 4above.

Preparation of Phase III

Phase III was formed with a 1:9 ratio (w/w) of Phase I to Phase II asdescribed above in EXAMPLE 4. The final concentration of loteprednoletabonate was 0.1% by weight of Phase III.

Analysis of the dispersion revealed that the median (d50) of theparticle size distribution was 159 nm, the mode was 160 nm, and 90% ofthe dispersed nano-structures were smaller than 303 nm. Thenanostructured dispersion was stable for at least 60 days at roomtemperature, with no settling or degradation observed. When examinedmicroscopically, the dispersion demonstrated an ordered microstructure,indicative of an ordered, but liquid-like state.

Additional analysis indicated that the amount of loteprednol etabonateencapsulated was 1.24 mg/G. The drug had an in vitro release of 35% in 3hours. Surprisingly, corneal permeability of the loteprednol etabonatewas 36% in 22 hours, as opposed to 8% for a commercially availableformulation of this drug, i.e., Lotemax® gel.

Example 6 Preparation of Drug-Free Nanostructured Dispersions by HighShear Mixing and Microfluidization

Preparation of Phase I

A 70 g amount of Phase I was prepared by mixing 7.01 g of MCT, 7.01 g ofcetyl alcohol, and 28 g of castor oil in a glass beaker. This mixturewas heated to 55° C. with continuous stirring on a hot plate to form ahomogenous mixture To this mixture, 7.07 g of polyethylene glycol-400,7.07 g of polypropylene glycol, and 4.97 g of PEG-40-stearate wereadded, followed by continued stirring at 55° C. After a homogenousmixture was formed, the heat component of the stir plate was switchedoff.

Preparation of Phase II

A 700 g amount of Phase II was prepared by mixing 0.21 g of tyloxapol,0.11 g of Tween 80, 1.441 g of citric acid monohydrate, 6.9688 g ofsodium citrate dehydrate, 0.140 g of Poloxamer 407, and 691.68 g of dH₂Owere added into a glass beaker. This mixture was stirred until the saltsare completely dissolved. The pH of Phase II was 6.0.

Preparation of Phase III

A total of 600 g of Phase III was formed as follows: 540 g of Phase IIwas maintained at 50-55° C. in a jacketed vessel connected to a chillerset at 50° C. A 60 g amount of Phase I, maintained at 50-55° C.; waspoured into Phase II and mixed for 15 min using a Silverson L5MA highshear mixer set at 7500 RPM. The resulting dispersion was passed 4 timesthrough a microfluidizer at a pressure of 28 psi.

Analysis of the dispersion revealed that the median (d50) of theparticle size distribution was 176 nm, the mode was 207 nm, and 90% ofthe dispersed nano-structures were smaller than 358 nm. Thenanostructured dispersion was stable over time at room temperature and,when examined microscopically, the dispersion demonstrated an orderedmicrostructure, indicative of an ordered, but liquid-like state. Themicrofluidization step resulted in a unimodal dispersion.

Example 7 Preparation of Drug-Free Nanostructured Dispersions by HighShear Mixing and Sonication

Preparation of Phase I

A 70 g amount of Phase I was prepared as described above in EXAMPLE 6.

Preparation of Phase II

A 70 g amount of Phase II was prepared also as described above inEXAMPLE 6.

Preparation of Phase III

A total of 600 g of Phase III was formed as follows: 540 g of Phase IIwas maintained at 40-45° C. in a jacketed vessel connected to a chiller.A 60 g amount of Phase I, maintained at 40-45° C.; was poured into PhaseII and mixed for 15 min. using a Silverson L5MA high shear mixer set at7500 RPM. The resulting dispersion was subjected to sonication with aHeilscher UP200S ultrasonic processor set to 50% amplitude using asonotrode S3 microtip. The dispersion was sonicated three times, and analiquot was taken after each time to monitor the effect of sonication.

Analysis of the dispersion revealed that the median (d50) of theparticle size distribution was 167 nm, the mode was 140 nm, and 75% ofthe dispersed nano-structures were smaller than 316 nm. Additionally,the nanostructured dispersion was stable over time at room temperatureand displayed an ordered microstructure when examined microscopically.

Example 8 Solubility of Fluticasone Propionate in NanostructuredDispersions

Phase I was prepared containing PEG-400 at 20%, PPG at 10%, cetylalcohol at 10%, MCT at 1%, PEG-stearate at 7%, poloxamer 407 at 2.22%,tyloxapol at 1%, polysorbate 80 at 1%, castor oil at 38%, andfluticasone propionate at 1-2%.

The composition of Phase II was sodium phosphate monobasic (0.017%),sodium phosphate dibasic (0.052%), sodium hyaluronate (0.15%), and dH₂O(99.78%).

Phase I was mixed with Phase II at a weight ratio of 1:9.

The concentration of fluticasone propionate achievable in thisformulation was 0.1-0.2%. Notably, when fluticasone propionate issolubilized separately in each excipient and the solubilizedcontribution from each excipient added, the maximum concentration offluticasone propionate expected is only 0.05%. Yet, the formulationsurprisingly solubilized fluticasone propionate at 0.1-0.2% by weight.

It was also determined that the combined presence of cetyl alcohol andMCT provided enhanced solubilization, even though the solubility offluticasone propionate in MCT is minimal (0.150 mg/mi). The solubilityof fluticasone propionate in cetyl alcohol was 0.3 mg/ml. As justdescribed, the final dispersion has a fluticasone propionateconcentration of 1-2 mg/ml (0.1-0.2%).

Not to be bound by theory, it is likely that the enhanced solubility ofthe drug results from the synergistic self-assembly of the phases toform an intercalated ordered phase.

Example 9 Effect of Hydrophobicity of Phase I

In this example, cholesterol was added to obtain a higher concentrationof hydrophobic excipients. The composition of Phase I was PEG-400 (20%),PPG (10%), cetyl alcohol (10%), cholesterol (2%), MCT (10%),PEG-stearate (5%), poloxamer 407 (2.22%), tyloxapol (1%), polysorbate 80(1%), castor oil (36.9%), and fluticasone propionate (1-5%).

The composition of Phase II was sodium phosphate monobasic (0.017%),sodium phosphate dibasic (0.052%), sodium hyaluronate (0.15%), and dH₂O(99.78%).

Phase I was mixed with Phase II at a weight ratio of 1:9.

The final concentration of fluticasone propionate achievable in thisformulation is 0.1-0.5% The maximum amount of fluticasone propionateexpected to be solubilized by adding up the amount solubilized in eachseparate excipient is 0.06%.

Again, in this EXAMPLE 9, a higher concentration of a hydrophobic drugwas achieved in the nano-dispersion than is theoretically possible fordrug solubilization by each individual component of Phase I

Example 10 In Vitro Drug Release from Nanodispersion

A nano-dispersion was produced using Phase I and Phase II describedabove in EXAMPLE 8 except that dexamethasone was incorporated instead offluticasone propionate. Phase I was mixed with Phase II with a Silversonhigh shear homogenizer, with both Phases at a temperature between 40-45°C., followed by high pressure homogenization using a microfluidizer. Themixture was passed through the microfluidizer 1 to 5 times at roomtemperature. The resultant mixture was then mixed overnight (18-22hours) with a Silverson high shear homogenizer at −10° C. After mixing,the dispersion was stored between 2-5° C. for 22-24 hours.

A micro-dispersion was formed of the identical components by mixingPhase I and Phase II with an overhead Scilogix stir-paddle mixer at 1800RPM for 2-3 hours. The temperature of each phase was 40-45° C. Themicro-dispersion was not incubated at 2-5° C. for 22-24 hours.

Particle size measurements revealed that the D50 of the nanodispersionwas between 100-250 nm while the D50 of the microdispersion was between60-90 μm.

In-vitro cumulative release rates of dexamethasone from thenanodispersion and the microdispersion were determined in phosphatebuffered saline, pH 7.4 at 37° C. The results indicated that 100% of thedexamethasone initially loaded in the microdispersion was released after40 h. By contrast, only ˜55% of the dexamethasone initially loaded inthe nanodispersion was released over the same time period. A cumulativerelease of 50% of the initial amount of dexamethasone was measured by 10h for the microdispersion as compared to 24 h for the nanodispersion.

The larger particles of the microdispersion have a larger diffusive pathlength for drug release. It was expected that the release from themicrodispersion would be slower than from the nanodispersion. As such,this result was unexpected.

Again, not to be bound by theory, it is thought that the intercalatedordered nanostructure of the nanodispersion creates tortuous diffusivepaths for the drug molecules to be released.

Example 11 Bioavailability Measured by Corneal Permeability

Four distinct formulations of fluticasone propionate (0.1%) wereprepared to ascertain the parameters leading to improvedbioavailability. Formulation I included all of the excipients listedabove in EXAMPLE 8 except for MCT, cetyl alcohol, and sodiumhyaluronate. Formulation II was the same as Formulation I but alsoincluded sodium hyaluronate. Formulation III was identical to thatdescribed in EXAMPLE 8. Formulation IV was identical to that describedin EXAMPLE 9.

The four formulations were prepared following the same mixing protocolsdescribed in EXAMPLE 8. The four formulations were tested for cornealpermeability as described above in EXAMPLE 2.

The results indicated that only 2.39% of the fluticasone propionate inFormulation I permeated through the cornea between 7-22 hours afterapplication of the formulation to the cornea. Similarly, only 2.3% ofthe fluticasone propionate in Formulation II diffused through the corneain 7-22 hours.

By contrast, 17% and 20% of the fluticasone propionate from FormulationIII and Formulation IV, respectively, diffused through the corneabetween 7 and 22 hours. Clearly, these two formulations demonstrated agreater bioavailability as compared to Formulations I and II, bothlacking MCT and cetyl alcohol.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, a person skilled in the art can easilyascertain the essential characteristics of the present disclosure, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the present disclosure to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

The invention claimed is:
 1. A method for producing a liquid crystallinedrug delivery system, the method comprising: forming a first solutioncontaining an active pharmaceutical ingredient (API) and a lipidiccomponent, the first solution being formed at a temperature of 37° C. to55° C.; cooling the first solution to a temperature of 30° C. to 45° C.;obtaining a second solution that includes a mucoadhesive hydrophilicpolymer and a buffer, the second solution being aqueous and maintainedat a temperature of −10° C. to 55° C.; mixing the first solution and thesecond solution to form a combined nano/micro-dispersion, the mixingaccomplished by ultrasonication, high shear homogenization, high shearmixing, high pressure homogenization, or a combination thereof; andcooling the nano/micro-dispersion to a temperature between 5° C. and 25°C. to form a liquid crystalline drug delivery system, wherein a weightratio between the first solution and the second solution is 1:1 to 1:15.2. The method of claim 1, wherein the lipidic component is selected fromthe group consisting of phosphatidylcholine, medium chain triglycerides,cetyl alcohol, cholesterol, polyethylene glycol (PEG)-stearate,cremophor, castor oil, mineral oil, and polysorbate 80, or a combinationthereof, and the mucoadhesive hydrophilic polymer is selected from thegroup consisting of sodium hyaluronate, xanthan gum, guar gum,hydroxypropyl methyl cellulose, carboxymethyl cellulose, polycarbopol,poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), 1-4beta glucan, tamarind seed polysaccharide, sodium alginate,polycarbopol, polyvinyl alcohol, and polycarbophil, or mixtures thereof.3. The method of claim 2, wherein the first solution further containsPEG-400, polypropylene glycol (PPG), poloxamer 407, tyloxapol, orpoly(lactic acid co-glycolic acid) and the API is an anti-inflammatory,a peptide, an anti-oxidant, an azole derivative, an anti-glaucoma drug,or a combination thereof.
 4. The method of claim 3, wherein the API isselected from the group consisting of fluticasone propionate,dexamethasone, betamethasone, budesonide, triamcinolone acetonide,methyl prednisolone, cortisone, beclometasone, fluticasone furoate,deoxycorticosterone acetate, loteprednol etabonate, difluprednate,fluorometholone, rimexolone, travoprost, moxifloxacin, and prednisoloneacetate, or a combination thereof.
 5. A liquid crystalline drug deliverysystem, comprising nanoparticles dispersed in an aqueous solution, thenanoparticles including a lipidic component, the aqueous solutioncontaining a mucoadhesive hydrophilic polymer and a buffer, wherein thelipidic component is selected from the group consisting ofphosphatidylcholine, medium chain triglycerides, cetyl alcohol,cholesterol, polyethylene glycol (PEG)-stearate, PEGylated castor oil,castor oil, and polysorbate 80, or a combination thereof, themucoadhesive hydrophilic polymer is selected from the group consistingof sodium hyaluronate, xanthan gum, guar gum, hydroxypropyl methylcellulose, 1-4 beta glucan, tamarind seed polysaccharide, sodiumalginate, polycarbopol, polyvinyl alcohol, and polycarbophil, orderivatives and mixtures thereof, and the nanoparticles have a size of40 nm to 900 nm.
 6. The liquid crystalline drug delivery system of claim5, wherein the nanoparticles further include PEG-400, polypropyleneglycol (PPG), poloxamer 407, tyloxapol, glycerol, castor oil,poly(lactic acid co-glycolic acid), or mixtures thereof.
 7. The liquidcrystalline drug delivery system of claim 5, wherein the nanoparticlesremain dispersed in the aqueous solution for at least 90 days.
 8. Theliquid crystalline drug delivery system of claim 5, further comprisingan active pharmaceutical ingredient (API) at 0.01-0.5% by weight of thesystem, wherein the API is loaded in the nanoparticles.
 9. The liquidcrystalline drug delivery system of claim 8, wherein the API is selectedfrom the group consisting of fluticasone propionate, dexamethasone,betamethasone, budesonide, triamcinolone acetonide, methyl prednisolone,cortisone, beclometasone, fluticasone furoate, deoxycorticosteroneacetate, loteprednol etabonate, difluprednate, fluorometholone,rimexolone, travoprost, moxifloxacin, posaconazole, prednisoloneacetate, nepafenac, bromfenac sodium, diclofenac, flurbiprofen sodium,ketorolac tromethamine, flurbiprofen sodium, tobramycin, netilmycin,erythromycin, bacitracin, azithromycin, gatifloxacin, gentamycinsulfate, levofloxacin, ofloxacin, sulfacetamide sodium, Polymyxin Bsulfate, sulfacetamide, neomycin sulfate, bacitracin zinc, gramicidin,or a combination thereof.
 10. The liquid crystalline drug deliverysystem of claim 8, wherein the API is a hydrophobic drug selected from aROCK inhibitor, an EGFR inhibitor, an A-1 agonist, a PARP inhibitor, anSOD mimetic, a PPAR agonist, a WNT inhibitor, a SYK-specific inhibitor,a JAK-specific inhibitor, a SYK/JAK or multi-kinase inhibitor, an MTOR,a STAT3 inhibitor, a VEGFR/PDGFR inhibitor, a c-Met inhibitor, an ALKinhibitor, an mTOR inhibitor, a PI3Kδ inhibitor, a PI3K/mTOR inhibitor,a p38/MAPK inhibitor, a macrolide, an azole derivative, a prostaglandin,an NO-releasing agent, a peptide, an NSAID, a steroid, an antibiotic, anantiviral, an antifungal, an anti-parasitic agent, a blood pressurelowering agent, an anti-neoplastic agent, an immunomodulatory drug, adiagnostic agent, or an anti-oxidant.
 11. The liquid crystalline drugdelivery system of claim 9, wherein the system has a pH of 6-7.5, anosmolarity of 250-340 mOsm/L, and a viscosity of 200-1000 cP.
 12. Theliquid crystalline drug delivery system of claim 11, wherein the API isfluticasone propionate present at 0.2% by weight of the system.
 13. Theliquid crystalline drug delivery system of claim 12, wherein thenanoparticles include, by weight of the system, 1% phosphatidylcholineand medium chain triglycerides, 1% cetyl alcohol, 2% PEG-400, 1% PPG,0.7% PEG-stearate, 0.22% poloxamer 407, 0.10% tyloxapol, 0.10%polysorbate 80, and 3.8% castor oil, and the aqueous solution contains,by weight of the system, 0.02% sodium phosphate monobasic, 0.05% sodiumphosphate dibasic, and 0.1-0.5% xanthan gum or 0.1-0.5%carboxymethylcellulose.
 14. The liquid crystalline drug delivery systemof claim 13, wherein the nanoparticles further include cholesterol at0.2% by weight of the system.
 15. A liquid crystalline drug deliverysystem for treating blepharitis, comprising, by weight, 0.01-0.2%fluticasone propionate or fluticasone furoate, 1% medium chaintriglycerides, 3.5-5% castor oil, 1.5-7% PEG-stearate, 0.5% cetylalcohol, 0.1-0.3% tyloxapol, 0.1-0.4% poloxamer 407, 1-5% PEG-400,0.154% monosodium phosphate, 0.21% disodium phosphate, 0.1-0.2% tween80, and 0.09-0.6% of a mucoadhesive polymer, wherein the system has a pHof 6-7.5, an osmolality of 250-340 mOsm/kg, and a viscosity of 200-1000cP.
 16. The liquid crystalline drug delivery system of claim 15, furthercomprising 0.05-0.5% moxifloxacin.
 17. A liquid crystalline dispersionfor treating glaucoma, comprising an anti-glaucoma drug, a mucoadhesivepolymer, and, by weight, 0.1-1% medium chain triglycerides, 4.5-5%castor oil, 1.5-7% PEG-stearate, 0.5% cetyl alcohol, 0.1-0.3% tyloxapol,0.1-0.4% poloxamer 407, 1-5% PEG-400, 0.154% monosodium phosphate, 0.21%disodium phosphate, and 0.1-0.2% tween 80, wherein the dispersion has apH of 6-7.5, an osmolality of 250-340 mOsm/kg, and a viscosity of200-1000 cP.
 18. An injectable composition for treating a posteriorsegment ocular disease, comprising a liquid crystalline dispersion thatincludes an API, a mucoadhesive polymer, polylactide-co-glycolide, and,by weight, 0.1-1% medium chain triglycerides, 1-5% castor oil, 0.1-0.7%PEG-stearate, 0.1-5% cetyl alcohol, 0.01-0.15% tyloxapol, 0.1-0.5%glyceryl stearate, 0.1-5% poloxamer 407, and 0.1-5% PEG-400.
 19. Amethod for treating blepharitis in a subject, the method comprisingidentifying a subject having blepharitis and administering to the eye ofthe subject the liquid crystalline drug delivery system of claim
 15. 20.The method of claim 19, wherein the liquid crystalline drug deliverysystem further comprises 0.05-0.5% moxifloxacin.
 21. A method fortreating glaucoma in a subject, the method comprising identifying asubject having glaucoma and administering to the eye of the subject theliquid crystalline dispersion of claim
 17. 22. The method of claim 21,wherein the liquid crystalline drug delivery system further comprisestravoprost.